Purification and some properties of inducible N-acetylglucosamine kinase from Candida albicans

Abstract

N-Acetylglucosamine kinase (ATP:2-acetamido-2-deoxy-d-glucose 6-phosphotransferase, EC 2.7.1.59) catalyzes the first reaction in the inducible N-acetylglucosamine catabolic pathway of Candida albicans, an obligatory aerobic yeast. As a part of continuing biochemical studies concerning the regulation of gene expression in a simple eukaryote, N-acetylglucosamine kinase has been purified and characterized biochemically. The enzyme has been purified about 300-fold from the crude extract and its molecular weight of 75 000 has been determined by Sephadex G-100 gel filtration. Isolation and analysis procedures are described. The kinase reaction is optimal within a pH range of 7-8. The enzyme is strictly specific for GlcNAc as phosphate acceptor; ATP is the phosphoryl group donor for the kinase reaction and to a lesser extent dATP and CTP. K_m values for GlcNAc and ATP are 1.33 mM and 1.82 mM, respectively. The enzyme requires Mg²⁺, which may be replaced by other bivalent metal ions such as Mn²⁺, Ca²⁺, Ba²⁺ and Co²⁺ for a lesser degree of effectiveness. The purified enzyme is extremely sensitive to thermal denaturation and becomes completely inactive by heating at 65°C for 2 min. The enzyme is also inactivated by sulphydryl reagents such as p-chloromercuribenzene sulfonic acid and N-ethylmaleimide.